The secretin receptor is prototypic of Family B G protein-coupled receptors, a group containing several important potential drug targets. The long-term goal of this work is to better understand the structure, function, and regulation of these receptors, gaining insights that will facilitate the development of new therapeutic strategies and new drugs that can act at a variety of targets within these molecules. The proposed projects are designed to test, extend, and refine our recently-proposed molecular model of the natural agonist-occupied secretin receptor and to elucidate the molecular basis of receptor activation and regulation. There are three broad aims for this proposal. The first aim is designed to acquire detailed structural insights into the global structure of the intact natural agonist-occupied secretin receptor, providing insights into the molecular basis for ligand docking and propagation of conformational changes from the amino-terminal domain to the core helical bundle domain. This will be investigated using directed photoaffinity labeling, including novel dual photolabile probes, as well as using immunological probes of accessible surfaces, and testing the evolving molecular models with targeted receptor mutagenesis. The second aim is designed to examine the cytosolic face of the secretin receptor, focusing on molecular determinants of coupling with G proteins, the configuration of G protein coupling to the physiologic quaternary dimeric structure of this receptor, and the impact of this on receptor function. This will be investigated using receptor mutagenesis to selectively disrupt G protein coupling, confirming the structural determinants by competition with peptides from the relevant regions. Photoaffinity labeling of the protomers involved in agonist docking and the G protein coupling will be used to explore the quaternary structure of affinity states associated with receptor homo-dimers. The third aim is designed to explore potential strategies for the development of receptor-active drugs, including agonists, antagonists, and allosteric modulators. This will be investigated using biochemical, molecular biological and pharmacological approaches to gain insights into the detailed structural determinants for orthosteric agonists and antagonists, as well as to define the possible allosteric site of action of the endogenous agonist sequence within the receptor amino terminus. Together, these efforts should provide the finest level of molecular detail available for understanding the structure and mechanisms of ligand binding, activation, and regulation of any receptor in this receptor family.